Cellular turnover of the hematopoietic system is supported by a small population of cells termed hematopoietic stem cells(HSCs). The mechanisms underlying the proliferation and differentiation of HSCs are incompletely understood. To study the cell kinetics of HSCs, the AC133+ cells, a subpopulation of hematopoietic stem and progenitor cells were first exposed to an expansion-promoting liquid medium, then grown in presence of either of the lineage specific cytokines EPO, G-CSF or TPO. Cell counts were obtained, and maturation was monitored by Wright-Giemas staining of cytospin smears. We observed a mean 290?17.3-, 312?42.7- and 15?3.1-fold increase in cell number on day 14 with EPO, G-CSF and TPO, respectively. For the evaluation of the proliferation and differentiation potential, after the 14 day growth, the cells were pelleted, washed and re-cultured in an alternate cytokine. Under these conditions, the original EPO-stimulated population contained ~90% granulocytes following a subsequent 10 day exposure to G-CSF, while the population from the original G-CSF-stimulated population grew ~95% late erythroid cells after a 14 day treatment with EPO. In contrast, the TPO-stimulated population showed no proliferation and differentiation with subsequent G-CSF or EPO incubation. To understand whether the persistence of progenitor cell population depends on the survival of quiescent cells or on the ability of cycling cells to self-renew and differentiate; AC133+ cells were grown at a concentration ~1x104 cells/ml in 0.9% methycellulose containing a cocktail of hematopoietic growth factors with EPO or G-CSF. When adequate clonal expansion was achieved, 20 individual colonies were isolated from EPO or G-CSF plates, randomly picked and pipeted into an alternate cytokine containing semi-solid medium. A mean of 8.8?9.2 myeloid colonies were identified from original erythroid, and of 2.5?3.5 erythriod colonies appeared in original myeloid cells on replating day 14.Stage-specific correlation of maturation with gene expression was conducted using single-cell RT-PCR and FISH. Analysis of the single cell expression patterns showed that erythroid and myeloid gene expression programs could be initiated prior to commitment to the erythroid or granulocytic lineages. These results indicate that stem and progenitor cells prime several different lineage-affiliated programs of gene activity prior to unilinerge commitment and differentiation. This system, with appropriate mathematical algorithms may be useful in investigating the "induced"versus "stochastic "models of HSC commitment.